Abstract
Calmodulin (CaM) regulation of voltage-gated calcium (Ca(V)1-2) channels is a powerful Ca(2+)-feedback mechanism to adjust channel activity in response to Ca(2+) influx. Despite progress in resolving mechanisms of CaM-Ca(V) feedback, the stoichiometry of CaM interaction with Ca(V) channels remains ambiguous. Functional studies that tethered CaM to Ca(V)1.2 suggested that a single CaM sufficed for Ca(2+) feedback, yet biochemical, FRET, and structural studies showed that multiple CaM molecules interact with distinct interfaces within channel cytosolic segments, suggesting that functional Ca(2+) regulation may be more nuanced. Resolving this ambiguity is critical as CaM is enriched in subcellular domains where Ca(V) channels reside, such as the cardiac dyad. We here localized multiple CaMs to the Ca(V) nanodomain by tethering either WT or mutant CaM that lack Ca(2+)-binding capacity to the pore-forming α-subunit of Ca(V)1.2, Ca(V)1.3, and Ca(V)2.1 and/or the auxiliary β(2A) subunit. We observed that a single CaM tethered to either the α or β(2A) subunit tunes Ca(2+) regulation of Ca(V) channels. However, when multiple CaMs are localized concurrently, Ca(V) channels preferentially respond to signaling from the α-subunit-tethered CaM. Mechanistically, the introduction of a second IQ domain to the Ca(V)1.3 carboxyl tail switched the apparent functional stoichiometry, permitting two CaMs to mediate functional regulation. In all, Ca(2+) feedback of Ca(V) channels depends exquisitely on a single CaM preassociated with the α-subunit carboxyl tail. Additional CaMs that colocalize with the channel complex are unable to trigger Ca(2+)-dependent feedback of channel gating but may support alternate regulatory functions.